bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1
2
3
4 Systemic administration of anti-CD20 indirectly reduces B cells in the inflamed meninges
5 in a chronic model of central nervous system autoimmunity
6
7 Yodit Tesfagiorgis1, Heather C Craig1, Kate A Parham1, and Steven M Kerfoot1*.
8
9
10 1 Department of Microbiology and Immunology
11 University of Western Ontario,
12 London, Ontario, Canada
13
14 * Corresponding Author:
15 mail: Dr. Steve Kerfoot, University of Western Ontario, Dental Sciences Building 3014, London,
16 Ontario, Canada, N6A 5C1
17 email: [email protected]
18 phone: 519 850 2329
19 fax: 519 661 3499
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20 Abstract:
21 Anti-CD20 B cell depleting therapies have demonstrated that B cells are important drivers of
22 disease progress in Multiple Sclerosis, although the pathogenic mechanisms are not well
23 understood. A population of B cells accumulates in the inflamed meninges in MS and also some
24 chronic animal models of disease, typically adjacent to demyelinating lesions. The role of these
25 meningeal B cells in disease is not known, nor is their susceptibility to anti-CD20 therapy. Here,
26 we administered anti-CD20 to 2D2 IgHMOG spontaneous experimental autoimmune
27 encephalomyelitis mice in the chronic phase of disease, after the establishment of meningeal B
28 cell clusters. Compared to the circulation, lymph nodes, and spleen, B cell depletion from the
29 CNS was delayed and not evident until 7d post administration of anti-CD20. Further, we did not
30 find evidence that anti-CD20 accessed meningeal B cells directly, but rather that depletion was
31 indirect and the result of ongoing turnover of the meningeal population and elimination of the
32 peripheral pool from which it is sustained. The reduction of B cell numbers in the CNS coincided
33 with less demyelination of the spinal cord white matter and also, surprisingly, an increase in the
34 number of T cells recruited to the meninges but not parenchyma.
35
36
37 Keywords: B cell, T cell, autoimmunity, EAE, MOG, Multiple Sclerosis, anti-CD20
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38 Introduction
39 Anti-CD20 B cell depleting therapies, which includes rituximab, ocrelizumab, and ofatumumab,
40 are effective in treating patients with relapsing(1–3) and progressive forms of multiple sclerosis
41 (MS) (4–6). In clinical trials anti-CD20 was shown to reduce the number of B cells in the
42 cerebral spinal fluid (CSF) (6–9) and peripheral blood (3, 10) and also results in a reduction of
43 important measures of disease activity including a reduction in new and existing brain lesions,
44 clinical relapses, disability progression (1–5, 10–12) and, importantly, T cell accumulation in the
45 CSF (9) and peripheral blood (8). The B cell lineage is best known for their production of
46 antibodies that target immune effector mechanisms to specific antigen targets. However, because
47 anti-CD20 therapies do not target antibody producing plasma cells, nor do they reduce antibody
48 levels within the therapeutic time frame (2, 8, 9), it is not clear how B cells are contributing to
49 disease pathogenesis.
50
51 B cells accumulate in the central nervous system (CNS) of MS patients, where they often form
52 clusters within the meninges directly adjacent to demyelinating lesions (13–16). Importantly,
53 these clusters have been shown to correlate to early onset of disease and severe cortical
54 pathology (14, 15). We and others have shown that this phenomenon is recapitulated in some
55 animal models of anti-myelin autoimmunity (experimental autoimmune encephalomyelitis -
56 EAE) (17–21). These clusters can become organized with separate T cell zones and B cell
57 follicles reminiscent of lymphoid tissue (13, 22), but more frequently they are disorganized
58 mixtures of B and T cells (23, 24). Because of their location within the inflamed CNS, it has
59 been hypothesized that these lymphoid clusters represent an environment where the autoimmune
60 response is propagated locally. Nevertheless, this has not yet been demonstrated and the
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61 pathogenic contributions to local disease remain unknown. Further, it is not yet known if B cells
62 in the meningeal clusters are susceptible to depletion with systemically administered anti-CD20
63 antibodies used to successfully treat MS.
64
65 B cells play multiple roles in MS and EAE, complicating the analysis of the therapeutic benefits
66 of B cell depletion. Work by ourselves and others suggests that, depending on the stage of
67 disease, both myelin-specific and non-specific B cells contribute different mechanisms to
68 disease. For instance, in a peptide induced model of EAE, depletion of all B cells, including non-
69 specific cells, prior to disease induction results in more severe disease (25, 26). This is attributed
70 to IL-10 production by B cells, demonstrating that at least some B cells can play a regulatory
71 role. In contrast, anti-myelin B cells contribute to disease initiation in models that incorporate B
72 cells in the autoimmune response. This includes 2D2 IgHMOG spontaneous EAE (sEAE) (27, 28)
73 and models induced by immunization with larger protein myelin antigens (26, 29, 30). Indeed,
74 EAE induced in mice by immunization with human myelin oligodendrocyte glycoprotein (MOG)
75 is completely dependent on B cells through the production of anti-MOG antibodies (31).
76 Nevertheless, we have shown that the B cell anti-myelin response is short-lived (32) and that
77 anti-myelin B cells are not found in meningeal clusters in the inflamed spinal cord in either
78 sEAE or MOG-protein induced EAE (33). Therefore, the role of anti-myelin-specific B cells
79 may be confined to the initiation of the disease, and thus the contribution of B cells to the
80 chronic phase of disease is likely from some other, non-myelin specific population and may
81 include those that accumulate in the CNS.
82
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83 Here, we set out to determine if B cells in meningeal clusters are susceptible to depletion by
84 systemic anti-mCD20 treatment. We specifically investigated B cell depletion following
85 treatment in the chronic phase of disease, after the establishment of B and T cell inflammation in
86 the CNS, in order to isolate the pathogenic contribution of these cells from any role B cells play
87 in disease initiation. Further, considering the association tween meningeal B cells and severe
88 disease, we wanted to determine if a reduction in these cells did correspond to a reduction in
89 pathology. To address these questions, we employed the 2D2 IgHMOG sEAE model which we
90 have shown results in a chronic disease course with consistent and robust, ongoing inflammation
91 in the spinal cord that includes B cell accumulation in the meninges adjacent to demyelinating
92 lesions (20). We found that systemic administration of anti-mCD20 does not target B cells in
93 meningeal clusters directly, but does reduce their numbers over time, likely due to the
94 elimination of peripheral B cells and the ongoing turnover of the meningeal population. Further,
95 we found that the reduction in B cells in meningeal clusters corresponds to reduced
96 demyelination in the adjacent lesion. Finally, and unexpectedly, B cell depletion also
97 corresponded to an increase in T cells specifically in the meninges, but not white matter.
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98 Materials and Methods
99 Mice:
100 Wild-type C57BL/6 and 2D2 TCR transgenic (211) were purchased from the Jackson
101 Laboratory. Mice expressing fluorescent dsRed (6051; Tg(CAG-DsRed∗MST)1Nagy/J) under
102 control of the β-Actin promoter within all nucleated cells were obtained from the Jackson
103 Laboratory. IgHMOG MOG-specific BCR knockin mice (212) were received as a gift from Dr. H.
104 Wekerle. All mice were housed under specific pathogen-free conditions at the West Valley
105 Barrier Facility at Western University Canada. Animal protocols were approved by the Western
106 University Animal Use Subcommittee.
107
108 Spontaneous 2D2 IgHMOG EAE model:
109 IgHMOG+/+ mice were crossed with 2D2+/- mice. Approximately 80% of IgHMOG+/- 2D2+/- double
110 mutant offspring spontaneously developed signs of EAE (sEAE) at 31 – 42d of age. Only mice
111 that developed signs of disease and met the conditions of chronic disease were included in
112 experiments. As both sexes develop chronic disease, both male and female mice were used in
113 experiments. Clinical disease was monitored daily with a modified 0-20 clinical scoring system
114 to evaluate tail paralysis, weakness and paralysis for each individual limb, and righting reflex.
115 Scores were determined as follows: Tails were scored as: 0, asymptomatic; 2, partial tail
116 paralysis; 4, complete tail paralysis. Each hind limb was scored as: 0, asymptomatic; 1, hind limb
117 weakness with a wobbly gate; 2, weight bearing but knuckling; 3, not weight bearing; 4,
118 complete hind limb paralysis. Each forelimb was scored as: 0, asymptomatic; 1, weak grasp yet
119 weight bearing; 2, not weight bearing; 3, complete forelimb paralysis. Finally, the righting reflex
120 was assessed as: 0, asymptomatic; 1, delayed righting reflex; 2, no righting reflex.
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121 Antibodies for flow cytometry and histology:
122 The following Abs were purchased from BD Bioscience: anti-CD4-v450 (RM4-5), anti-CD45R-
123 A647 (RA3-6B2), anti-CD138-BV421 (281-2), CD19-BV711 (1D3) and mIgG2a-biotin (R19-
124 15). The following Abs were purchased from BioLegend: anti-CD4-A647 (RM4-5), anti-CD3-
125 A488 (17A2), CD45R-APC-Cy7 (RA3-6B2), anti-IgKappa-biotin (RMK-12), anti-Ly6G-A647
126 (1A8), anti-rabbit-DyLight555 (Poly4064), anti-CD19-A488 (6D5) and anti-GFAP-A488
127 (2E1.E9). Streptavidin-APC, Streptavidin-eF570 and anti-CD4-PECy5 (RM4-5) was purchased
128 from eBioscience. Anti-Myelin Basic Protein (MBP)-rabbit polyclonal antibody and anti-Ki67
129 (SP6) unconjugated was purchased from Abcam.
130
131 Anti-mCD20 B cell depletion:
132 Anti-mouse-CD20 (5D2), the murine surrogate of rituximab was received as a generous gift from
133 Genentech, South San Francisco, USA. 150μg of the drug was administered i.v. (unless
134 otherwise stated) during the chronic phase of disease, ~2 weeks post disease onset. Anti-IgG2a
135 (MG2a-53) acquired from BioLegend was used as an isotype control and injected at the same
136 concentration i.v. Mice were monitored for EAE disease severity for either 2- or 7-days
137 following treatment before collecting the blood, lymph node, spleen and spinal cord for flow
138 cytometry or histology.
139
140 Flow cytometry:
141 The blood, lymph nodes (inguinal, axillary, and cervical), spleen and the spinal cord were
142 harvested from mice for flow cytometry analysis as previously described (18, 33). Briefly, blood
143 was isolated through a cardiac puncture with needles pre-washed with 0.5M EDTA, after which
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144 the mouse was perfused with ice cold PBS prior to harvesting other tissues. To isolate
145 inflammatory cells from spinal cords, individual spinal cords were dissociated through a wire
146 mesh after which leukocytes were isolated using a Percoll (GE Healthcare Life Sciences)
147 gradient, collecting leukocytes at the 37/90% Percoll interface. Cell suspensions were generated
148 from lymphoid tissues by first dissociating them between frosted glass slides. The spleen and
149 blood were then lysed for 2 min at 37°C to remove red blood cells with ACK buffer. Dead cells
150 were identified by staining with a Fixable Viability Dye eFluor506 (eBioscience) according to
151 manufacturer’s protocol. All cells were then blocked with an anti-Fcγ receptor, CD16/32 2.4G2
152 (BD biosciences), in PBS containing 2% FBS for 30 min on ice. Cells were then stained on ice
153 for 30 mins with the listed combination of staining Abs, followed by a secondary stain with
154 streptavidin for 15 mins on ice where necessary. Spleen cells were fixed in 2% PFA in PBS prior
155 to running cells to prevent cell clumping. Flow cytometry was performed on a BD
156 Immunocytometry Systems LSRII cytometer. Analysis was then completed using FlowJo
157 software (TreeStar).
158
159 Immunofluorescent histology:
160 Spinal cord and lymph node tissue were prepared for histology as previously described (20).
161 Briefly, at the end of the experiment or earlier, if mice reached a predetermined endpoint lymph
162 node sections and spinal cord tissue spanning the cervical to lumbar regions were isolated and
163 fixed in PLP. Spinal cords were then cut into five to nine evenly spaced sections and frozen in
164 OCT (TissueTek) media. Tissue was then cut in serial cryostat sections at 7μm. Prior to staining,
165 all slide-mounted tissue sections were blocked with PBS containing 1% BSA, 0.1% Tween-20,
166 and 10% rat serum. After staining, sections were mounted with ProLong Gold Antifade Reagent
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167 (Invitrogen). When staining for MBP, tissue sections were additionally pre-treated to remove
168 lipids in a series of ethanol gradients (0%, 50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, 50%
169 and 0% ethanol in deionized water for one minute at each concentration) prior to the blocking
170 step. Tiled images of whole spinal cord sections were collected using a DM5500B fluorescence
171 microscope (Leica) at 20x.
172
173 mMOGtag immunization and cell isolation for transfer:
174 To induce activated anti-MOG T and B cells for subsequent transfer into chronic sEAE mice,
175 2D2 FRP+ mice were immunized with a fusion protein antigen based on the extracellular domain
176 of mouse MOG protein (mMOGtag) (18, 34) as previously described (33). Briefly, 6-8 week old
+ 177 2D2 FRP mice were immunized with 0.5 mg of mMOGtag in CFA (Sigma-Aldrich) s.c. at two
178 sites near the base of the tail. 5d post immunization, draining inguinal lymph nodes were
179 harvested and dissociated into transfer buffer (10 mM HEPES, 25 µg/mL gentamycin, 2.5%
180 Acid citrate dextrose solution A in PBS). Suspended and filtered cells were injected i.v. into the
181 tail vein of chronic sEAE mice at a 1:1 ratio of donor to recipient mice.
182
183 Imaging and Statistical analysis:
184 Microscopy images were analyzed using Fiji software. The number of infiltrating B and T cells
185 were determined by cell counting the number of cells per spinal cord area. The total area of the
186 spinal cord was determined by either FluoroMyelin staining or glial fibrillary acidic protein
187 (GFAP) staining, while regions of demyelination were determined by the absence of MBP. The
188 percent of demyelination was calculated by dividing the area of demyelination to the total area of
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189 the spinal cord. To determine the size of the cluster area, I looked at regions of the meninges
190 (determined by the lack of GFAP staining) that contained B and T cell infiltration.
191 PRISM software (GraphPad, La Jolla, California) was used for all statistical analysis. A Student t
192 test was used for single comparisons, and ANOVA followed by a Student t test with Bonferroni
193 correction was used for multiple comparisons.
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194 Results
195 CNS-infiltrating B cells are reduced 7d post i.v. treatment with anti-mCD20
196 To determine the optimal dose of anti-mCD20 (5D2) to effectively deplete B cells in mice, we
197 administered either 150μg or 250μg (based on previously published studies (35)) of anti-mCD20
198 (5D2) into wild type mice i.v. B cell numbers were analyzed in the blood, peripheral lymph
199 nodes and spleen by flow cytometry 1d post treatment. As expected, B cells were almost entirely
200 undetectable in the blood (Figure 1A), demonstrating that depletion of B cells in the circulation
201 is rapid and effective. B cell numbers in lymph nodes (axial, brachial and inguinal), and the
202 spleen were also reduced, but not to the same degree as in the blood. This suggests that anti-
203 mCD20 that is administered i.v. has limited access to B cells in tissues compared to cells in the
204 circulation, consistent with previous analysis of i.p. administered anti-mCD20 (26). As no
205 difference was observed between the two doses, all subsequent experiments used 150μg as the
206 treatment dose.
207
208 To determine if systemic depletion of B cells also reduces the number of B cells in the inflamed
209 spinal cord in sEAE, we administered anti-mCD20 i.v. during the chronic phase of disease
210 (~2wks post onset, Figure 1B, see Table 1 for the disease duration, severity, and sex of
211 experimental mice). We have shown that T cell infiltration of the parenchyma and white matter
212 demyelination is ongoing in this model at this timepoint, and also that B cells have established
213 clusters in the meninges of the spinal cord (20). We analyzed B cell numbers in the blood, lymph
214 nodes, spleen, and spinal cord by flow cytometry 2- or 7-days following treatment. As expected
215 based on the above pilot study in healthy mice, B cells in the circulating blood were almost
216 undetectable 2d post treatment and remained absent 7d post treatment, signifying that B cells
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217 were not replaced by hematopoiesis within this timeframe (Figure 1C). No decrease in CD4 or
218 CD8 T cells were observed, as expected (data not shown). B cells were also significantly
219 reduced in lymphoid tissues (lymph nodes and spleen) 2d and 7d post anti-mCD20-treatment
220 compared to mice treated with isotype control antibody, but to a lesser extent than in the
221 circulation (Figure 1C). In contrast, there was little indication of a reduction in B cells in the
222 inflamed spinal cord by 2d pos treatment and it was not until 7d post treatment that a significant
223 reduction in B cells was observed, both as a percentage of live cells and absolute number of B
224 cells. This delay in the depletion of B cells in the inflamed CNS could be because it takes longer
225 for the anti-mCD20 antibody to cross the blood brain barrier, or it could be because there is
226 ongoing turnover of the CNS B cell population, and the elimination of circulating cells prevents
227 the recruitment of replacement cells to the inflamed CNS.
228
229 Anti-mCD20 administration reduces the number of B cells in the meninges and this
230 corresponds with less demyelination
231 Because meningeal B cell clusters correlate with disease severity in MS (14, 15) and EAE (19,
232 20), we performed additional experiments to determine if systemic B cell depletion reduces B
233 cell accumulation in the meninges and also spinal cord pathology in chronic sEAE. sEAE mice
234 in the chronic phase of disease were administered anti-mCD20 i.v. and spinal cords were
235 harvested 7d later for histological analysis (see Table 2 for disease parameters). Initial analysis
236 revealed no differences in any measured parameters between isotype control treated mice and
237 untreated mice, and therefore both were included in the control group.
238
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239 As we observed previously (20), clusters of B cells were evident in the spinal cord meninges of
240 chronic sEAE mice, and these clusters typically formed adjacent to regions of T cell infiltration
241 into the spinal cord white matter and regions of demyelination (Figure 2A). Most clusters were a
242 mixture of B cells and T cells, however in some circumstances the largest clusters showed
243 evidence of organized separation of these cell types (as shown in Figure 2A). Examples of
244 organized and disorganized clusters could be found in both control and anti-mCD20-treated
245 mice. However, and consistent with the reduction in CNS B cells observed by flow cytometry
246 (Figure 1C) above, treatment significantly reduced the density of B cells within meningeal
247 clusters compared to untreated controls (Figure 2B). Absolute numbers of meningeal B cells
248 were also lower, but this did not reach significance. Interestingly, the area of the clusters
249 themselves did not change over this time period (Figure 2C). Importantly, the area of white
250 matter demyelination was significantly reduced in treated mice (Figure 2D, E). This did not
251 translate to a significant difference in disease score within this 7d timeframe (Figure 2F),
252 consistent with recent observations by others (36) who similarly observed improved tissue
253 pathology after a longer-duration treatment with anti-CD20 that did not translate into a
254 measurable change in disease severity using standard scoring systems. Nevertheless, our
255 observations suggest that meningeal B cells contribute to local tissue pathology.
256
257 Reductions in meningeal B cells are not likely the result of direct anti-mCD20-mediated
258 depletion
259 In the above experiments we established that depletion of B cells in the spinal cord following
260 systemic administration of anti-mCD20 is delayed relative to that observed in the circulation and
261 lymphatic tissues (Figure 1C). To determine if i.v. administered anti-mCD20 has differential
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262 access to B cells in the inflamed spinal cord compared to B cells in the circulating blood and
263 lymphatic tissue, we used flow cytometry to identify B cells bound by i.v. administered 5D2 anti-
264 mCD20 2 or 7d post treatment. Of the very few detectable B220+ CD19+ B cells remaining in the
265 circulation 2d post treatment, approximately half were bound by 5D2 (Figure 3A). This suggests
266 that B cells bound by 5D2 are rapidly eliminated in the blood, perhaps as they rejoin the
267 circulation from peripheral tissues. In lymphoid tissue (spleen and lymph nodes), where
268 measurable numbers of B cells were still evident (Figure 1C), the large majority of B cells were
269 bound by 5D2 by d2 and remained so at d7. This may indicate that anti-mCD20-mediated
270 depletion within tissues is slower than it is in the blood. In contrast, almost none of the B cells in
271 the spinal cord were bound by 5D2 2d after i.v. administration, and this only increased to ~10%
272 of CNS B cells 7d post administration. This suggests that 5D2 may not be able to cross the blood
273 brain barrier to label cells already in the tissue, or that access to the CNS is much slower than
274 other tissues.
275
276 In order to differentiate between these possibilities, we performed a separate experiment to look
277 by histology for direct evidence of B cells bound by 5D2 in the meninges, and also to determine
278 the pattern of binding within meningeal clusters. If systemically administered anti-mCD20 does
279 cross the blood brain barrier on its own by 7d post administration to directly bind B cells in
280 meningeal clusters, we would expect to see all or most B cells within a given cluster to be evenly
281 bound by the 5D2 antibody. This is not what we observed, however. Instead, 5D2-bound B cells
282 were most commonly observed in meningeal clusters as individual, brightly-stained cells
283 distributed among unlabeled cells (Figure 3B, panels i, iii, and iv). This pattern is more
284 consistent with B cells being heavily labelled in the circulation and then carrying the anti-
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285 mCD20 antibody with them as they are recruited to the cluster. Interestingly, accumulation of
286 5D2-bound B cells was highly variable within individual clusters, where some clusters did not
287 appear to have any (Figure 3B, panels ii and v) and, in rarer instances, most B cells were bound
288 by 5D2 (panel iv). This inconsistency suggests that B cell recruitment and turnover rates differ
289 between different clusters. Indeed, different patterns could be observed between clusters from the
290 same tissue section (compare panel i to ii, and iv to v), and the degree of 5D2 binding
291 corresponded to the degree of B cell elimination from the cluster. This suggests that anti-CD20
292 treatment may effectively eliminate B cells in “active” clusters with ongoing recruitment.
293 Regardless, these observations together suggest that B cells encounter and are bound by
294 systemically administered anti-CD20 in the circulation and are then recruited to the inflamed
295 CNS, rather than the antibody itself crossing the blood meningeal barrier and binding cells for
296 depletion directly in the tissue.
297
298 T cells accumulate in the meninges following anti-mCD20 depletion of B cells
299 As noted above, the area of the meningeal lymphoid clusters did not change following anti-
300 mCD20 treatment (Figure 2C), despite the fact that the density of B cells within the clusters was
301 reduced (Figure 2B). Further analysis revealed that, with the decrease in B cells, CD4 T cells
302 increased in the meninges of treated mice, resulting in a significant increase in both the numbers
303 and density of these cells within clusters compared to control treated mice (Figure 4 A and B).
304 Overall, this resulted in a significant shift in the dominant cell type within clusters from B cells
305 to T cells (Figure 4C). This increase in CD4+ T cells is not likely due to increased proliferation,
306 as there was no difference in the percentage of Ki67+ T cells as measured by microscopy (Figure
307 4D) or flow cytometry (data not shown). Flow cytometry also failed to show a difference in
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308 expression of IL-10, IFNγ, or IL-17 by CNS T cells (data not shown), again suggesting that the
309 reduction in meningeal B cells did not impact T cell activation by this timepoint. Further, there
310 was no change in the number of CD4+ T cells in the spinal cord white matter by d7 post
311 treatment (Figure 4E), indicating that T cells do not leave the parenchyma to populate the
312 meninges.
313
314 Finally, we transferred lymph node cells from MOG-immunized 2D2 RFP+ mice (5d post
315 immunization) into chronic sEAE mice 7d post treatment with anti-mCD20. Spinal cords were
316 harvested 4d post transfer to look for evidence of recruitment of the transferred RFP+
317 lymphocytes to the spinal cord. Interestingly, fewer RFP+ B cells were found in the lymph nodes
318 of anti-mCD20-treated mice compared to untreated controls (flow cytometry data not shown),
319 indicating that depletion of peripheral B cells was still effective 7-11d post treatment. RFP+
320 CD4+ cells, but not RFP+ CD45R+ B cells were readily apparent in meningeal clusters of anti-
321 mCD20-treated mice by histology (Figure 4F), suggesting that extensive T cell recruitment to
322 the inflamed CNS is ongoing following B cell depletion and that recruitment from the circulation
323 is the likely source of the greater numbers of cluster T cells.
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324 Discussion
325 Here, we characterized the effects of anti-mCD20 depletion during the chronic phase of sEAE in
326 2D2 IgHMOG mice. We found that B cell depletion in the inflamed spinal cord was delayed
327 compared to peripheral tissues, including circulating blood, lymph nodes, and spleen. We
328 attribute this delay to the mechanism of reduction of meningeal B cell numbers, which we
329 believe results from the ongoing turnover of the population and the removal of the peripheral B
330 cell pool from which it is replenished. Indeed, previous work from our lab shows that B cells are
331 continuously recruited to meningeal clusters from the circulation at the chronic phase of EAE
332 (33). Further, we did not find evidence that the 5D2 antibody crossed the blood brain barrier on
333 its own, and our observations are more consistent with a scenario where B cells are bound by
334 anti-CD20 in the periphery and carry the antibody with them as they are recruited into the
335 inflamed CNS. Regardless, we consistently found that systemic administration of anti-mCD20
336 did ultimately result in fewer B cells in the CNS and specifically in the meninges of treated
337 compared to control mice.
338
339 By focusing on the chronic phase of sEAE we show that systemically administered, anti-CD20 B
340 cell-targeting therapies do reduce B cell numbers within already established lymphoid clusters in
341 the inflamed CNS. Most previous studies investigating the effects of B cell depletion in models
342 of CNS autoimmunity focused on the initiation and acute phase of disease. Treatment at the
343 acute phase of disease or earlier would prevent the formation of meningeal clusters, and may also
344 target other B cell populations with different roles in disease. Indeed, it is clear that depending on
345 the model, B cell depletion before or shortly after disease induction results in less or more severe
346 disease. For example, depleting B cells prior to disease onset in MOG35-55 peptide induced EAE
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347 results in increased disease severity (25, 26, 35) which is attributed to the loss of IL-10
348 production by B cell lineage cells (25). In contrast, when B cells are depleted prior to induction
349 of EAE using larger protein antigens, disease severity is often reduced (26, 29, 30, 35, 37).
350 Further, when B cells are depleted during the acute phase of recombinant MOG1-117 EAE (4d
351 post EAE onset), frequencies of Th1, Th17 and regulatory T cells in the peripheral blood and
352 CNS were decreased 14d post treatment (26), suggesting a role for B cells in modulating T cell
353 responses in this early phase of disease, most likely from outside of the CNS.
354
355 Clusters of B cells in the meninges are associated with more severe disease in both humans (14,
356 15) and our sEAE model (20), and it follows that their reduction through anti-CD20 treatment
357 coincided with a reduction in white matter demyelination in our studies. It is not clear if this is
358 because demyelination was prevented from occurring in the first place, or if there was some
359 opportunity for remyelination within the 7d treatment window. Remyelination following more
360 extended anti-CD20 treatment in a different chronic model of EAE was observed by Breakell
361 et.al. (36). The difference in white matter pathology following only 7d of treatment in our studies
362 indicates that the therapeutic benefit of B cell depletion can be rapid, and that it is temporally
363 associated with the reduction of B cells within established clusters in the meninges.
364 Nevertheless, we cannot exclude the possibility that therapeutic benefit was through some other
365 peripheral population. Still, unlike B cell depletion in the acute phase of disease discussed above,
366 we did not observe any evidence of changes in cytokine production by T cells in our chronic
367 model.
368
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369 We were surprised to find that, while systemic administration of anti-CD20 did reduce the
370 number of B cells in meningeal clusters, it also resulted in an increase in the number of T cells
371 within the meninges. Typically, increased T cell infiltration of the CNS is associated with worse
372 tissue pathology and disease. Further, we did not find evidence that the T cells in the meninges
373 were less activated or more regulatory, based on levels of proliferation and cytokine production.
374 Still, it must be emphasized that the increase in T cell numbers was limited to the meninges, as
375 we observed no change in numbers of parenchymal, white matter T cells following anti-mCD20
376 treatment. Further, while we did observe a small number of the transferred T cells in white
377 matter lesions of anti-mCD20-treated chronic sEAE mice, the vast majority remained confined to
378 the meninges. We think that it is likely that, given time, the overall number of infiltrating T cells
379 will go down, including those in the meninges. This would be in line with observations in human
380 MS where B cell depletion is associated with less CNS inflammation over the months following
381 treatment (1–5, 10, 11).
382
383 The identification of meningeal inflammatory foci in both secondary progressive (13–15) and
384 primary progressive MS (38, 39) led to the hypothesis that the pathogenic autoimmune response
385 is propagated from these locations. As stated, meningeal lesions have been correlated to
386 accelerated disease severity (15), and increased axonal atrophy in the brain and spinal cord (16,
387 40), suggesting they are a deleterious site of inflammation. Here, we show in a mouse model that
388 recapitulates these features of disease that these B cell clusters are susceptible to intervention
389 through peripheral administration of anti-CD20. Further, we show that a reduction in these cells
390 is associated with improved pathology, further supporting the hypothesis that they contribute
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391 pathologic mechanisms to local inflammation. Future studies will be required to determine the
392 mechanistic action(s) of meningeal B cells in chronic CNS pathology.
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393 Acknowledgements:
394 The authors would like to thank the veterinarians and animal care staff at the West Valley Barrier
395 Facility for their excellent husbandry of our experimental animals. YT is the recipient of an
396 endMS Doctoral Studentships from the Multiple Sclerosis Society of Canada (MSSOC). KAP is
397 the recipient of an endMS Post-Doctoral Fellowship from MSSOC. This study was funded by
398 operating grants from the Canadian Institutes of Health Research and the MSSOC.
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399 Table 1: Phenotype of mice treated in flow cytometry experiments.
Average STDEV n
age of onset 29.89 3.14
d of onset 15.11 5.95 Isotype max score 15.67 0.71 9 Control end score 14.33 1.66
F (%) 55.56
age of onset 28.44 2.88
d of onset 14.44 5.90
d2 5D2 Rx max score 14.89 1.27 9
end score 12.67 2.24
F (%) 55.56
age of onset 32.67 1.86
d of onset 17.83 5.95
d7 5D2 Rx max score 14.83 1.33 6
end score 11.67 3.20
F (%) 33.33
400
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401 Table 2: Phenotype of mice treated in immunofluorescence experiments.
Average STDEV n
age of onset 29.25 3.105295
d of onset 17.5 4.14
Control max score 15.3 0.71 8
end score 13.8 1.75
F (%) 50
age of onset 27.8 3.63
d of onset 22.2 1.30 d7 5D2 max score 15.2 0.84 5 Rx end score 10.8 2.28
F (%) 40
402
23 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
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27 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
544 Figure 1. Depletion of meningeal B cells by systemically administered anti-mCD20 is
545 delayed compared to other tissues. (A) Wild type mice were administered saline or either
546 150μg or 250μg anti-mCD20 (5D2) i.v. 1d post injection, blood, lymph nodes (axial, brachial
547 and inguinal), and the spleen were harvested for flow cytometry analysis of B cell numbers. (B)
548 2D2 IgHMOG sEAE mice in the chronic phase disease were treated with anti-mCD20 antibodies
549 or isotype control. Tissue was harvested 2d or 7d later for analysis by flow cytometry. (C) Data
550 is represented as percentage of live cells or absolute number of cells, as indicated. Each symbol
551 represents and individual mouse. *p <0.05, **** p <0.0001.
552
553
554 Figure 2: Anti-mCD20 treatment reduces meningeal B cell infiltration and demylination by
555 7d post treatment. (A) Representative immunofluorescence images of spinal cords from an
556 isotype-control treated mouse (left) and d7 anti-mCD20 treated mouse (right). These examples
557 were chosen to represent especially large B cell clusters, although the majority were smaller and
558 showed less evidence of separation between B cells and T cells (see Figures 3 and 4 for
559 additional examples). In the case of the control spinal cord (left), extensive T cell infiltration and
560 demyelination of the adjacent white matter is apparent. (B) The number and density of B cells in
561 the meninges, as well as total cluster area (C - defined by the presence of B and T cells), was
562 determined from multiple sections from each mouse. (D) Representative images showing
563 extensive demyelination in a control sEAE mouse (top) and less demyelination in a 5D2-treated
564 sEAE mouse (bottom). (E) Area of demyelination was determined by measuring regions staining
565 with GFAP but not MBP and expressed as a percentage of the total area of the spinal cord
566 section. Each symbol represents an individual mouse. *p <0.05, **p <0.01. (F) Disease severity
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
567 of control and anti-mCD20 treated mice used in the histological analysis above was tracked
568 (left), along with the relative change in disease score over the final 7d prior to sacrifice (right).
569 Data is shown as mean +/- SEM.
570
571
572 Figure 3: Meningeal B cell depletion is indirect and is likely the result of eliminating
573 circulating cells. 2D2 IgHMOG sEAE mice were administered isotype antibody or anti-mCD20
574 (5D2). (A) Circulating blood, lymph nodes (axial, brachial and inguinal), spleen and spinal cord
575 were harvested 2d or 7d post treatment for analysis by flow cytometry. Anti-IgG2a was used to
576 identify cells bound by 5D2. Each symbol represents an individual mouse. *p <0.05, **p <0.01,
577 **** p <0.0001. (B) Representative immunofluorescence images of spinal cord meningeal B cell
578 clusters a single chronic sEAE mouse (of 3) d7 post treatment with anti-mCD20 showing
579 different patterns of 5D2 binding of cluster B cells. Panels i and ii are different clusters from the
580 same section from the cervical spinal cord, and panels iv and v are different clusters from the
581 same section from the thoracic spinal cord.
582
583
584 Figure 4: T cell infiltration of the meninges increases following B cell depletion. (A) A
585 representative immunofluorescence image of a spinal cord from an isotype-control treated mouse
586 (top) and a d7 anti-mCD20 (5D2) depleted mouse (bottom) showing a change in the dominant
587 meningeal infiltrates from B cells to T cells. CD4+ cells and CD45R+ cells in the meninges were
588 counted and the area of meningeal clusters was measured to determine the number and density of
589 T cells in the clusters (B) and to calculate the ratio between B cells and T cells in the meninges
29 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
590 (C). Separate sections were stained with Ki67 and CD4 to determine the percentage of CD4+ T
591 cells in cell cycle (D). CD4+ T cells in the spinal cord parenchyma were counted (E). Each
592 symbol represents an individual mouse. *p <0.05, **p <0.01. (F) sEAE mice in the chronic
593 phase of disease were treated with anti-mCD20. 7d later, cells were isolated from the draining
594 lymph nodes of RFP+ 2D2 mice that had been immunized with MOG protein 5d prior. These
595 cells were transferred i.v. into the treated sEAE mice. 4d post transfer, spinal cords were
596 harvested for histological imaging of transferred RFP+ cells into the inflamed spinal cords.
30 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427667; this version posted January 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.